Genetic inhibition by double-stranded RNA

ABSTRACT

A process is provided of introducing an RNA into a living cell to inhibit gene expression of a target gene in that cell. The process may be practiced ex vivo or in vivo. The RNA has a region with double-stranded structure. Inhibition is sequence-specific in that the nucleotide sequences of the duplex region of the RNA and of a portion of the target gene are identical. The present invention is distinguished from prior art interference in gene expression by antisense or triple-strand methods.

RELATED APPLICATION

[0001] This application claims the benefit of U.S. Provisional Appln.No. 60/068,562, filed Dec. 23, 1997.

GOVERNMENT RIGHTS

[0002] This invention was made with U.S. government support under grantnumbers GM-37706, GM-17164, HD-33769 and GM-07231 awarded by theNational Institutes of Health. The U.S. government has certain rights inthe invention.

BACKGROUND OF THE INVENTION

[0003] 1. Field of the Invention

[0004] The present invention relates to gene-specific inhibition of geneexpression by double-stranded ribonucleic acid (dsRNA).

[0005] 2. Description of the Related Art

[0006] Targeted inhibition of gene expression has been a long-felt needin biotechnology and genetic engineering. Although a major investment ofeffort has been made to achieve this goal, a more comprehensive solutionto this problem was still needed.

[0007] Classical genetic techniques have been used to isolate mutantorganisms with reduced expression of selected genes. Although valuable,such techniques require laborious mutagenesis and screening programs,are limited to organisms in which genetic manipulation is wellestablished (e.g., the existence of selectable markers, the ability tocontrol genetic segregation and sexual reproduction), and are limited toapplications in which a large number of cells or organisms can besacrificed to isolate the desired mutation. Even under thesecircumstances, classical genetic techniques can fail to producemutations in specific target genes of interest, particularly whencomplex genetic pathways are involved. Many applications of moleculargenetics require the ability to go beyond classical genetic screeningtechniques and efficiently produce a directed change in gene expressionin a specified group of cells or organisms. Some such applications areknowledge-based projects in which it is of importance to understand whateffects the loss of a specific gene product (or products) will have onthe behavior of the cell or organism. Other applications are engineeringbased, for example: cases in which is important to produce a populationof cells or organisms in which a specific gene product (or products) hasbeen reduced or removed. A further class of applications istherapeutically based in which it would be valuable for a functioningorganism (e.g., a human) to reduce or remove the amount of a specifiedgene product (or products). Another class of applications provides adisease model in which a physiological function in a living organism isgenetically manipulated to reduce or remove a specific gene product (orproducts) without making a permanent change in the organism's genome.

[0008] In the last few years, advances in nucleic acid chemistry andgene transfer have inspired new approaches to engineer specificinterference with gene expression. These approaches are described below.

[0009] Use of Antisense Nucleic Acids to Engineer Interference

[0010] Antisense technology has been the most commonly describedapproach in protocols to achieve gene-specific interference. Forantisense strategies, stochiometric amounts of single-stranded nucleicacid complementary to the messenger RNA for the gene of interest areintroduced into the cell. Some difficulties with antisense-basedapproaches relate to delivery, stability, and dose requirements. Ingeneral, cells do not have an uptake mechanism for single-strandednucleic acids, hence uptake of unmodified single-stranded material isextremely inefficient. While waiting for uptake into cells, thesingle-stranded material is subject to degradation. Because antisenseinterference requires that the interfering material accumulate at arelatively high concentration (at or above the concentration ofendogenous mRNA), the amount required to be delivered is a majorconstraint on efficacy. As a consequence, much of the effort indeveloping antisense technology has been focused on the production ofmodified nucleic acids that are both stable to nuclease digestion andable to diffuse readily into cells. The use of antisense interferencefor gene therapy or other whole-organism applications has been limitedby the large amounts of oligonucleotide that need to be synthesized fromnon-natural analogs, the cost of such synthesis, and the difficulty evenwith high doses of maintaining a sufficiently concentrated and uniformpool of interfering material in each cell.

[0011] Triple-Helix Approaches to Engineer Interference

[0012] A second, proposed method for engineered interference is based ona triple helical nucleic acid structure. This approach relies on therare ability of certain nucleic acid populations to adopt atriple-stranded structure. Under physiological conditions, nucleic acidsare virtually all single- or double-stranded, and rarely if ever formtriple-stranded structures. It has been known for some time, however,that certain simple purine- or pyrimidine-rich sequences could form atriple-stranded molecule in vitro under extreme conditions of pH (i.e.,in a test tube). Such structures are generally very transient underphysiological conditions, so that simple delivery of unmodified nucleicacids designed to produce triple-strand structures does not yieldinterference. As with antisense, development of triple-strand technologyfor use in vivo has focused on the development of modified nucleic acidsthat would be more stable and more readily absorbed by cells in vivo. Anadditional goal in developing this technology has been to producemodified nucleic acids for which the formation of triple-strandedmaterial proceeds effectively at physiological pH.

[0013] Co-Suppression Phenomena and Their Use in Genetic Engineering

[0014] A third approach to gene-specific interference is a set ofoperational procedures grouped under the name “co-suppression”. Thisapproach was first described in plants and refers to the ability oftransgenes to cause silencing of an unlinked but homologous gene. Morerecently, phenomena similar to co-suppression have been reported in twoanimals: C. elegans and Drosophila. Co-suppression was first observed byaccident, with reports coming from groups using transgenes in attemptsto achieve over-expression of a potentially useful locus. In some casesthe over-expression was successful while, in many others, the result wasopposite from that expected. In those cases, the transgenic plantsactually showed less expression of the endogenous gene. Severalmechanisms have so far been proposed for transgene-mediatedco-suppression in plants; all of these mechanistic proposals remainhypothetical, and no definitive mechanistic description of the processhas been presented. The models that have been proposed to explainco-suppression can be placed in two different categories. In one set ofproposals, a direct physical interaction at the DNA- or chromatin-levelbetween two different chromosomal sites has been hypothesized to occur;an as-yet-unidentified mechanism would then lead to de novo methylationand subsequent suppression of gene expression. Alternatively, some havepostulated an RNA intermediate, synthesized at the transgene locus,which might then act to produce interference with the endogenous gene.The characteristics of the interfering RNA, as well as the nature of theinterference process, have not been determined. Recently, a set ofexperiments with RNA viruses have provided some support for thepossibility of RNA intermediates in the interference process. In theseexperiments, a replicating RNA virus is modified to include a segmentfrom a gene of interest. This modified virus is then tested for itsability to interfere with expression of the endogenous gene. Initialresults with this technique have been encouraging, however, theproperties of the viral RNA that are responsible for interferenceeffects have not been determined and, in any case, would be limited toplants which are hosts of the plant virus.

[0015] Distinction Between the Present Invention and AntisenseApproaches

[0016] The present invention differs from antisense-mediatedinterference in both approach and effectiveness. Antisense-mediatedgenetic interference methods have a major challenge: delivery to thecell interior of specific single-stranded nucleic acid molecules at aconcentration that is equal to or greater than the concentration ofendogenous mRNA. Double-stranded RNA-mediated inhibition has advantagesboth in the stability of the material to be delivered and theconcentration required for effective inhibition. Below, we disclose thatin the model organism C. elegans, the present invention is at least100-fold more effective than an equivalent antisense approach (i.e.,dsRNA is at least 100-fold more effective than the injection of purifiedantisense RNA in reducing gene expression). These comparisons alsodemonstrate that inhibition by double-stranded RNA must occur by amechanism distinct from antisense interference.

[0017] Distinction Between the Present Invention and Triple-HelixApproaches

[0018] The limited data on triple strand formation argues against theinvolvement of a stable triple-strand intermediate in the presentinvention. Triple-strand structures occur rarely, if at all, underphysiological conditions and are limited to very unusual base sequencewith long runs of purines and pyrimidines. By contrast, dsRNA-mediatedinhibition occurs efficiently under physiological conditions, and occurswith a wide variety of inhibitory and target nucleotide sequences. Thepresent invention has been used to inhibit expression of 18 differentgenes, providing phenocopies of null mutations in these genes of knownfunction. The extreme environmental and sequence constraints ontriple-helix formation make it unlikely that dsRNA-mediated inhibitionin C. elegans is mediated by a triple-strand structure.

[0019] Distinction Between Present Invention and Co-SuppressionApproaches

[0020] The transgene-mediated genetic interference phenomenon calledco-suppression may include a wide variety of different processes. Fromthe viewpoint of application to other types of organisms, theco-suppression phenomenon in plants is difficult to extend. Aconfounding aspect in creating a general technique based onco-suppression is that some transgenes in plants lead to suppression ofthe endogenous locus and some do not. Results in C. elegans andDrosophila indicate that certain transgenes can cause interference(i.e., a quantitative decrease in the activity of the correspondingendogenous locus) but that most transgenes do not produce such aneffect. The lack of a predictable effect in plants, nematodes, andinsects greatly limits the usefulness of simply adding transgenes to thegenome to interfere with gene expression. Viral-mediated co-suppressionin plants appears to be quite effective, but has a number of drawbacks.First, it is not clear what aspects of the viral structure are criticalfor the observed interference. Extension to another system would requirediscovery of a virus in that system which would have these properties,and such a library of useful viral agents are not available for manyorganisms. Second, the use of a replicating virus within an organism toeffect genetic changes (e.g., long- or short-term gene therapy) requiresconsiderably more monitoring and oversight for deleterious effects thanthe use of a defined nucleic acid as in the present invention.

[0021] The present invention avoids the disadvantages of thepreviously-described methods for genetic interference. Severaladvantages of the present invention are discussed below, but numerousothers will be apparent to one of ordinary skill in the biotechnologyand genetic engineering arts.

SUMMARY OF THE INVENTION

[0022] A process is provided for inhibiting expression of a target genein a cell. The process comprises introduction of RNA with partial orfully double-stranded character into the cell or into the extracellularenvironment. Inhibition is specific in that a nucleotide sequence from aportion of the target gene is chosen to produce inhibitory RNA. Wedisclose that this process is (1) effective in producing inhibition ofgene expression, (2) specific to the targeted gene, and (3) general inallowing inhibition of many different types of target gene.

[0023] The target gene may be a gene derived from the cell, anendogenous gene, a transgene, or a gene of a pathogen which is presentin the cell after infection thereof. Depending on the particular targetgene and the dose of double stranded RNA material delivered, theprocedure may provide partial or complete loss of function for thetarget gene. A reduction or loss of gene expression in at least 99% oftargeted cells has been shown. Lower doses of injected material andlonger times after administration of dsRNA may result in inhibition in asmaller fraction of cells. Quantitation of gene expression in a cell mayshow similar amounts of inhibition at the level of accumulation oftarget mRNA or translation of target protein.

[0024] The RNA may comprise one or more strands of polymerizedribonucleotide; it may include modifications to either thephosphate-sugar backbone or the nucleoside. The double-strandedstructure may be formed by a single self-complementary RNA strand or twocomplementary RNA strands. RNA duplex formation may be initiated eitherinside or outside the cell. The RNA may be introduced in an amount whichallows delivery of at least one copy per cell. Higher doses ofdouble-stranded material may yield more effective inhibition. Inhibitionis sequence-specific in that nucleotide sequences corresponding. to theduplex region of the RNA are targeted for genetic inhibition. RNAcontaining a nucleotide sequences identical to a portion of the targetgene is preferred for inhibition. RNA sequences with insertions,deletions, and single point mutations relative to the target sequencehave also been found to be effective for inhibition. Thus, sequenceidentity may optimized by alignment algorithms known in the art andcalculating the percent difference between the nucleotide sequences.Alternatively, the duplex region of the RNA may be defined functionallyas a nucleotide sequence that is capable of hybridizing with a portionof the target gene transcript.

[0025] The cell with the target gene may be derived from or contained inany organism (e.g., plant, animal, protozoan, virus, bacterium, orfungus). RNA may be synthesized either in vivo or in vitro. EndogenousRNA polymerase of the cell may mediate transcription in vivo, or clonedRNA polymerase can be used for transcription in vivo or in vitro. Fortranscription from a transgene in vivo or an expression construct, aregulatory region may be used to transcribe the RNA strand (or strands).

[0026] The RNA may be directly introduced into the cell (i.e.,intracelltularly); or introduced extracellularly into a cavity,interstitial space, into the circulation of an organism, introducedorally, or may be introduced by bathing an organism in a solutioncontaining RNA. Methods for oral introduction include direct mixing ofRNA with food of the organism, as well as engineered approaches in whicha species that is used as food is engineered to express an RNA, then fedto the organism to be affected. Physical methods of introducing nucleicacids include injection directly into the cell or extra-cellularinjection into the organism of an RNA solution.

[0027] The advantages of the present invention include: the ease ofintroducing double-stranded RNA into cells, the low concentration of RNAwhich can be used, the stability of double-stranded RNA, and theeffectiveness of the inhibition. The ability to use a low concentrationof a naturally-occurring nucleic acid avoids several disadvantages ofanti-sense interference. This invention is not limited to in vitro useor to specific sequence compositions, as are techniques based ontriple-strand formation. And unlike antisense interference,triple-strand interference, and co-suppression, this invention does notsuffer from being limited to a particular set of target genes, aparticular portion of the target gene's nucleotide sequence, or aparticular transgene or viral delivery method. These concerns have beena serious obstacle to designing general strategies according to theprior art for inhibiting gene expression of a target gene of interest.

[0028] Furthermore, genetic manipulation becomes possible in organismsthat are not classical genetic models. Breeding and screening programsmay be accelerated by the ability to rapidly assay the consequences of aspecific, targeted gene disruption. Gene disruptions may be used todiscover the function of the target gene, to produce disease models inwhich the target gene are involved in causing or preventing apathological condition, and to produce organisms with improved economicproperties.

BRIEF DESCRIPTION OF THE DRAWINGS

[0029]FIG. 1 shows the genes used to study RNA-mediated geneticinhibition in C. elegans. Intron-exon structure for genes used to testRNA-mediated inhibition are shown (exons: filled boxes; introns: openboxes; 5′ and 3′ untranslated regions: shaded; unc-22⁹,unc-54¹²,fem-1¹⁴, and hlh-1¹⁵).

[0030] FIGS. 2 A-I show analysis of inhibitory RNA effects in individualcells. These experiments were carried out in a reporter strain (calledPD4251) expressing two different reporter proteins, nuclear GFP-LacZ andmitochondrial GFP. The micrographs show progeny of injected animalsvisualized by a fluorescence microscope. Panels A (young larva), B(adult), and C (adult body wall; high magnification) result frominjection of a control RNA (ds-unc22A). Panels D-F show progeny ofanimals injected with ds-gfpG. Panels G-I demonstrate specificity.Animals are injected with ds-lacZL RNA, which should affect the nuclearbut not the mitochondrial reporter construct. Panel H shows a typicaladult, with nuclear GFP-LacZ lacking in almost all body-wall muscles butretained in vulval muscles. Scale bars are 20 μm.

[0031] FIGS. 3 A-D show effects of double-stranded RNA corresponding tomex-3 on levels of the endogenous mRNA. Micrographs show in situhybridization to embryos (dark stain). Panel A: Negative control showinglack of staining in the absence of hybridization probe. Panel B: Embryofrom uninjected parent (normal pattern of endogenous mex-3 RNA²⁰). PanelC: Embryo from a parent injected with purified mex-3B antisense RNA.These embryos and the parent animals retain the mex-3 mRNA, althoughlevels may have been somewhat less than wild type. Panel D: Embryo froma parent injected with dsRNA corresponding to mex-3B; no mex-3 RNA wasdetected. Scale: each embryo is approximately 50 μm in length.

[0032]FIG. 4 shows inhibitory activity of unc-22A as a function ofstructure and concentration. The main graph indicates fractions in eachbehavioral class. Embryos in the uterus and already covered with aneggshell at the time of injection were not affected and, thus, are notincluded. Progeny cohort groups are labeled 1 for 0-6 hours, 2 for 6-15hours, 3 for 15-27 hours, 4 for 27-41 hours, and 5 for 41-56 hours Thebottom-left diagram shows genetically derived relationship betweenunc-22 gene dosage and behavior based on analyses of unc-22heterozygotes and polyploids^(8,3).

[0033] FIGS. 5 A-C show examples of genetic inhibition followingingestion by C. elegans of dsRNAs from expressing bacteria. Panel A:General strategy for production of dsRNA by cloning a segment ofinterest between flanking copies of the bacteriophage T7 promoter andtranscribing both strands of the segment by transfecting a bacterialstrain (BL21/DE3)²⁸ expressing the T7 polymerase gene from an inducible(Lac) promoter. Panel B: A GFP-expressing C. elegans strain, PD4251 (seeFIG. 2), fed on a native bacterial host. Panel C: PD4251 animals rearedon a diet of bacteria expressing dsRNA corresponding to the codingregion for gfp.

DETAILED DESCRIPTION OF THE INVENTION

[0034] The present invention provides a method of producingsequence-specific inhibition of gene expression by introducingdouble-stranded RNA (dsRNA). A process is provided for inhibitingexpression of a target gene in a cell. The process comprisesintroduction of RNA with partial or fully double-stranded character intothe cell. Inhibition is sequence-specific in that a nucleotide sequencefrom a portion of the target gene is chosen to produce inhibitory RNA.We disclose that this process is (1) effective in producing inhibitionof gene expression, (2) specific to the targeted gene, and (3) generalin allowing inhibition of many different types of target gene.

[0035] The target gene may be a gene derived from the cell (i.e., acellular gene), an endogenous gene (i.e., a cellular gene present in thegenome), a transgene (i.e., a gene construct inserted at an ectopic sitein the genome of the cell), or a gene from a pathogen which is capableof infecting an organism from which the cell is derived. Depending onthe particular target gene and the dose of double stranded RNA materialdelivered, this process may provide partial or complete loss of functionfor the target gene. A reduction or loss of gene expression in at least99% of targeted cells has been shown.

[0036] Inhibition of gene expression refers to the absence (orobservable decrease) in the level of protein and/or mRNA product from atarget gene. Specificity refers to the ability to inhibit the targetgene without manifest effects on other genes of the cell. Theconsequences of inhibition can be confirmed by examination of theoutward properties of the cell or organism (as presented below in theexamples) or by biochemical techniques such as RNA solutionhybridization, nuclease protection, Northern hybridization, reversetranscription, gene expression monitoring with a microarray, antibodybinding, enzyme linked immunosorbent assay (ELISA), Western blotting,radioimmunoassay (RIA), other immunoassays, and fluorescence activatedcell analysis (FACS). For RNA-mediated inhibition in a cell line orwhole organism, gene expression is conveniently assayed by use of areporter or drug resistance gene whose protein product is easilyassayed. Such reporter genes include acetohydroxyacid synthase (AHAS),alkaline phosphatase (AP), beta galactosidase (LacZ), beta glucoronidase(GUS), chloramphenicol acetyltransferase (CAT), green fluorescentprotein (GFP), horseradish peroxidase (HRP), luciferase (Luc), nopalinesynthase (NOS), octopine synthase (OCS), and derivatives thereof.Multiple selectable markers are available that confer resistance toampicillin, bleomycin, chloramphenicol, gentamycin, hygromycin,kanamycin, lincomycin, methotrexate, phosphinothricin, puromycin, andtetracyclin.

[0037] Depending on the assay, quantitation of the amount of geneexpression allows one to determine a degree of inhibition which isgreater than 10%, 33%, 50%, 90%, 95% or 99% as compared to a cell nottreated according to the present invention. Lower doses of injectedmaterial and longer times after administration of dsRNA may result ininhibition in a smaller fraction of cells (e.g., at least 10%, 20%, 50%,75%, 90%, or 95% of targeted cells). Quantitation of gene expression ina cell may show similar amounts of inhibition at the level ofaccumulation of target mRNA or translation of target protein. As anexample, the efficiency of inhibition may be determined by assessing theamount of gene product in the cell: mRNA may be detected with ahybridization probe having a nucleotide sequence outside the region usedfor the inhibitory double-stranded RNA, or translated polypeptide may bedetected with an antibody raised against the polypeptide sequence ofthat region.

[0038] The RNA may comprise one or more strands of polymerizedribonucleotide. It may include modifications to either thephosphate-sugar backbone or the nucleoside. For example, thephosphodiester linkages of natural RNA may be modified to include atleast one of a nitrogen or sulfur heteroatom. Modifications in RNAstructure may be tailored to allow specific genetic inhibition whileavoiding a general panic response in some organisms which is generatedby dsRNA. Likewise, bases may be modified to block the activity ofadenosine deaminase. RNA may be produced enzymatically or bypartiautotal organic synthesis, any modified ribonucleotide can beintroduced by in vitro enzymatic or organic synthesis.

[0039] The double-stranded structure may be formed by a singleself-complementary RNA strand or two complementary RNA strands. RNAduplex formation may be initiated either inside or outside the cell. TheRNA may be introduced in an amount which allows delivery of at least onecopy per cell. Higher doses (e.g., at least 5, 10, 100, 500 or 1000copies per cell) of double-stranded material may yield more effectiveinhibition; lower doses may also be useful for specific applications.Inhibition is sequence-specific in that nucleotide sequencescorresponding to the duplex region of the RNA are targeted for geneticinhibition.

[0040] RNA containing a nucleotide sequences identical to a portion ofthe target gene are preferred for inhibition. RNA sequences withinsertions, deletions, and single point mutations relative to the targetsequence have also been found to be effective for inhibition. Thus,sequence identity may optimized by sequence comparison and alignmentalgorithms known in the art (see Gribskov and Devereux, SequenceAnalysis Primer, Stockton Press, 1991, and references cited therein) andcalculating the percent difference between the nucleotide sequences by,for example, the Smith-Waterman algorithm as implemented in the BESTFITsoftware program using default parameters (e.g., University of WisconsinGenetic Computing Group). Greater than 90% sequence identity, or even100% sequence identity, between the inhibitory RNA and the portion ofthe target gene is preferred. Alternatively, the duplex region of theRNA may be defined functionally as a nucleotide sequence that is capableof hybridizing with a portion of the target gene transcript (e.g., 400mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, 50° C. or 70° C. hybridizationfor 12-16 hours; followed by washing). The length of the identicalnucleotide sequences may be at least 25, 50, 100, 200, 300 or 400 bases.

[0041] As disclosed herein, 100% sequence identity between the RNA andthe target gene is not required to practice the present invention. Thusthe invention has the advantage of being able to tolerate sequencevariations that might be expected due to genetic mutation, strainpolymorphism, or evolutionary divergence.

[0042] The cell with the target gene may be derived from or contained inany organism. The organism may a plant, animal, protozoan, bacterium,virus, or fungus. The plant may be a monocot, dicot or gymnosperm; theanimal may be a vertebrate or invertebrate. Preferred microbes are thoseused in agriculture or by industry, and those that are pathogenic forplants or animals. Fungi include organisms in both the mold and yeastmorphologies.

[0043] Plants include arabidopsis; field crops (e.g., alfalfa, barley,bean, corn, cotton, flax, pea, rape, rice, rye, safflower, sorghum,soybean, sunflower, tobacco, and wheat); vegetable crops (e.g.,asparagus, beet, broccoli, cabbage, carrot, cauliflower, celery,cucumber, eggplant, lettuce, onion, pepper, potato, pumpkin, radish,spinach, squash, taro, tomato, and zucchini); fruit and nut crops (e.g.,almond, apple, apricot, banana, blackberry, blueberry, cacao, cherry,coconut, cranberry, date, fajoa, filbert, grape, grapefruit, guava,kiwi, lemon, lime, mango, melon, nectarine, orange, papaya, passionfruit, peach, peanut, pear, pineapple, pistachio, plum, raspberry,strawberry, tangerine, walnut, and watermelon); and ornamentals (e.g.,alder, ash, aspen, azalea, birch, boxwood, camellia, carnation,chrysanthemum, elm, fir, ivy, jasmine, juniper, oak, palm, poplar, pine,redwood, rhododendron, rose, and rubber).

[0044] Examples of vertebrate animals include fish, mammal, cattle,goat, pig, sheep, rodent, hamster, mouse, rat, primate, and human;invertebrate animals include nematodes, other worms, drosophila, andother insects. Representative generae of nematodes include those thatinfect animals (e.g., Ancylostoma, Ascaridia, Ascaris, Bunostomum,Caenorhabditis, Capillaria, Chabertia, Cooperia, Dictyocaulus,Haemonchus, Heterakis, Nematodirus, Oesophagostomum, Ostertagia,Oxyuris, Parascaris, Strongylus, Toxascaris, Trichuris,Trichostrongylus, Tfhchonema, Toxocara, Uncinaria) and those that infectplants (e.g., Bursaphalenchus, Criconemella, Diiylenchus, Ditylenchus,Globodera, Helicotylenchus, Heterodera, Longidorus, Melodoigyne,Nacobbus, Paratylenchus, Pratylenchus, Radopholus, Rotelynchus,Tylenchus, and Xiphinema). Representative orders of insects includeColeoptera, Diptera, Lepidoptera, and Homoptera.

[0045] The cell having the target gene may be from the germ line orsomatic, totipotent or pluripotent, dividing or non-dividing, parenchymaor epithelium, immortalized or transformed, or the like. The cell may bea stem cell or a differentiated cell. Cell types that are differentiatedinclude adipocytes, fibroblasts, myocytes, cardiomyocytes, endothelium,neurons, glia, blood cells, megakaryocytes, lymphocytes, macrophages,neutrophils, eosinophils, basophils, mast cells, leukocytes,granulocytes, keratinocytes, chondrocytes, osteoblasts, osteoclasts,hepatocytes, and cells of the endocrine or exocrine glands.

[0046] RNA may be synthesized either in vivo or in vitro. Endogenous RNApolymerase of the cell may mediate transcription in vivo, or cloned RNApolymerase can be used for transcription in vivo or in vitro. Fortranscription from a transgene in vivo or an expression construct, aregulatory region (e.g., promoter, enhancer, silencer, splice donor andacceptor, polyadenylation) may be used to transcribe the RNA strand (orstrands). Inhibition may be targeted by specific transcription in anorgan, tissue, or cell type; stimulation of an environmental condition(e.g., infection, stress, temperature, chemical inducers); and/orengineering transcription at a developmental stage or age. The RNAstrands may or may not be polyadenylated; the RNA strands may or may notbe capable of being translated into a polypeptide by a cell'stranslational apparatus. RNA may be chemically or enzymaticallysynthesized by manual or automated reactions. The RNA may be synthesizedby a cellular RNA polymerase or a bacteriophage RNA polymerase (e.g.,T3, T7, SP6). The use and production of an expression construct areknown in the art^(32, 33, 34) (see also WO 97/32016; U.S. Pat. Nos.5,593,874, 5,698,425, 5,712,135, 5,789,214, and 5,804,693; and thereferences cited therein). If synthesized chemically or by in vitroenzymatic synthesis, the RNA may be purified prior to introduction intothe cell. For example, RNA can be purified from a mixture by extractionwith a solvent or resin, precipitation, electrophoresis, chromatography,or a combination thereof. Alternatively, the RNA may be used with no ora minimum of purification to avoid losses due to sample processing. TheRNA may be dried for storage or dissolved in an aqueous solution. Thesolution may contain buffers or salts to promote annealing, and/orstabilization of the duplex strands.

[0047] RNA may be directly introduced into the cell (i.e.,intracellularly); or introduced extracellularly into a cavity,interstitial space, into the circulation of an organism, introducedorally, or may be introduced by bathing an organism in a solutioncontaining the RNA. Methods for oral introduction include direct mixingof the RNA with food of the organism, as well as engineered approachesin which a species that is used as food is engineered to express theRNA, then fed to the organism to be affected. For example, the RNA maybe sprayed onto a plant or a plant may be genetically engineered toexpress the RNA in an amount sufficient to kill some or all of apathogen known to infect the plant. Physical methods of introducingnucleic acids, for example, injection directly into the cell orextracellular injection into the organism, may also be used. We discloseherein that in C. elegans, double-stranded RNA introduced outside thecell inhibits gene expression. Vascular or extravascular circulation,the blood or lymph system, the phloem, the roots, and the cerebrospinalfluid are sites where the RNA may be introduced. A transgenic organismthat expresses RNA from a recombinant construct may be produced byintroducing the construct into a zygote, an embryonic stem cell, oranother multipotent cell derived from the appropriate organism.

[0048] Physical methods of introducing nucleic acids include injectionof a solution containing the RNA, bombardment by particles covered bythe RNA, soaking the cell or organism in a solution of the RNA, orelectroporation of cell membranes in the presence of the RNA. A viralconstruct packaged into a viral particle would accomplish both efficientintroduction of an expression construct into the cell and transcriptionof RNA encoded by the expression construct. Other methods known in theart for introducing nucleic acids to cells may be used, such aslipid-mediated carrier transport, chemical-mediated transport, such ascalcium phosphate, and the like. Thus the RNA may be introduced alongwith components that perform one or more of the following activities:enhance RNA uptake by the cell, promote annealing of the duplex strands,stabilize the annealed strands, or other-wise increase inhibition of thetarget gene.

[0049] The present invention may be used to introduce RNA into a cellfor the treatment or prevention of disease. For example, dsRNA may beintroduced into a cancerous cell or tumor and thereby inhibit geneexpression of a gene required for maintenance of thecarcinogenic/tumorigenic phenotype. To prevent a disease or otherpathology, a target gene may be selected which is required forinitiation or maintenance of the disease/pathology. Treatment wouldinclude amelioration of any symptom associated with the disease orclinical indication associated with the pathology.

[0050] A gene derived from any pathogen may be targeted for inhibition.For example, the gene could cause immunosuppression of the host directlyor be essential for replication of the pathogen, transmission of thepathogen, or maintenance of the infection. The inhibitory RNA could beintroduced in cells in vitro or ex vivo and then subsequently placedinto an animal to affect therapy, or directly treated by in vivoadministration. A method of gene therapy can be envisioned. For example,cells at risk for infection by a pathogen or already infected cells,particularly human immunodeficiency virus (HIV) infections, may betargeted for treatment by introduction of RNA according to theinvention. The target gene might be a pathogen or host gene responsiblefor entry of a pathogen into its host, drug metabolism by the pathogenor host, replication or integration of the pathogen's genome,establishment or spread of an infection in the host, or assembly of thenext generation of pathogen. Methods of prophylaxis (i.e., prevention ordecreased risk of infection), as well as reduction in the frequency orseverity of symptoms associated with infection, can be envisioned.

[0051] The present invention could be used for treatment or developmentof treatments for cancers of any type, including solid tumors andleukemias, including: apudoma, choristoma, branchioma, malignantcarcinoid syndrome, carcinoid heart disease, carcinoma (e.g., Walker,basal cell, basosquamous, Brown-Pearce, ductal, Ehrlich tumor, in situ,Krebs 2, Merkel cell, mucinous, non-small cell lung, oat cell,papillary, scirrhous, bronchiolar, bronchogenic, squamous cell, andtransitional cell), histiocytic disorders, leukemia (e.g., B cell, mixedcell, null cell, T cell, T-cell chronic, HTLV-II-associated, lymphocyticacute, lymphocytic chronic, mast cell, and myeloid), histiocytosismalignant, Hodgkin disease, immunoproliferative small, non-Hodgkinlymphoma, plasmacytoma, reticuloendotheliosis, melanoma,chondroblastoma, chondroma, chondrosarcoma, fibroma, fibrosarcoma, giantcell tumors, histiocytoma, lipoma, liposarcoma, mesothelioma, myxoma,myxosarcoma, osteoma, osteosarcoma, Ewing sarcoma, synovioma,adenofibroma, adenolymphoma, carcinosarcoma, chordoma,cranio-pharyngioma, dysgerminoma, hamartoma, mesenchymoma, mesonephroma,myosarcoma, ameloblastoma, cementoma, odontoma, teratoma, thymoma,trophoblastic tumor, adenocarcinoma, adenoma, cholangioma,cholesteatoma, cylindroma, cystadenocarcinoma, cystadenoma, granulosacell tumor, gynandroblastoma, hepatoma, hidradenoma, islet cell tumor,Leydig cell tumor, papilloma, Sertoli cell tumor, theca cell tumor,leiomyoma, leiomyosarcoma, myoblastoma, myoma, myosarcoma, rhabdomyoma,rhabdomyosarcoma, sarcoma, ependymoma, ganglioneuroma, glioma,medulloblastoma, meningioma, neurilemmoma, neuroblastoma,neuroepithelioma, neurofibroma, neuroma, paraganglioma, paragangliomanonchromaffin, angiokeratoma, angiolymphoid hyperplasia witheosinophilia, angioma sclerosing, angiomatosis, glomangioma,hemangioendothelioma, hemangioma, hemangiopericytoma, hemangiosarcoma,lymphangioma, lymphangiomyoma, lymphangiosarcoma, pinealoma,carcinosarcoma, chondrosarcoma, cystosarcoma phyllodes, fibrosarcoma,hemangiosarcoma, leiomyosarcoma, leukosarcoma, liposarcoma,lymphangiosarcoma, myosarcoma, myxosarcoma, ovarian carcinoma,rhabdomyosarcoma, sarcoma (e.g., Ewing, experimental, Kaposi, and mastcell), neoplasms (e.g., bone, breast, digestive system, colorectal,liver, pancreatic, pituitary, testicular, orbital, head and neck,central nervous system, acoustic, pelvic, respiratory tract, andurogenital), neurofibromatosis, and cervical dysplasia, and fortreatment of other conditions in which cells have become immortalized ortransformed. The invention could be used in combination with othertreatment modalities, such as chemotherapy, cryotherapy, hyperthermia,radiation therapy, and the like.

[0052] As disclosed herein, the present invention may is not limited toany type of target gene or nucleotide sequence. But the followingclasses of possible target genes are listed for illustrative purposes:developmental genes (e.g., adhesion molecules, cyclin kinase inhibitors,Wnt family members, Pax family members, Winged helix family members, Hoxfamily members, cytokines/lymphokines and their receptors,growth/differentiation factors and their receptors, neurotransmittersand their receptors); oncogenes (e.g., ABL1, BCL1, BCL2, BCL6, CBFA2,CBL, CSF1R, ERBA, ERBB, EBRB2, ETS1, ETS1, ETV6, FGR, FOS, FYN, HCR,HRAS, JUN, KRAS, LCK, LYN, MDM2, MLL, MYB, MYC, MYCL1, MYCN, NRAS, PIM1,PML, RET, SRC, TAL1, TCL3, and YES); tumor suppressor genes (e.g., APC,BRCA1, BRCA2, MADH4, MCC, NF1, NF2, RB1, TP53, and WT1); and enzymes(e.g., ACC synthases and oxidases, ACP desaturases and hydroxylases,ADP-glucose pyrophorylases, ATPases, alcohol dehydrogenases, amylases,amyloglucosidases, catalases, cellulases, chalcone synthases,chitinases, cyclooxygenases, decarboxylases, dextrinases, DNA and RNApolymerases, galactosidases, glucanases, glucose oxidases, granule-boundstarch synthases, GTPases, helicases, hemicellulases, integrases,inulinases, invertases, isomerases, kinases, lactases, lipases,lipoxygenases, lysozymes, nopaline synthases, octopine synthases,pectinesterases, peroxidases, phosphatases, phospholipases,phosphorylases, phytases, plant growth regulator synthases,polygalacturonases, proteinases and peptidases, pullanases,recombinases, reverse transcriptases, RUBISCOs, topoisomerases, andxylanases).

[0053] The present invention could comprise a method for producingplants with reduced susceptibility to climatic injury, susceptibility toinsect damage, susceptibility to infection by a pathogen, or alteredfruit ripening characteristics. The targeted gene may be an enzyme, aplant structural protein, a gene involved in pathogenesis, or an enzymethat is involved in the production of a non-proteinaceous part of theplant (i.e., a carbohydrate or lipid). If an expression construct isused to transcribe the RNA in a plant, transcription by a wound- orstress-inducible; tissue-specific (e.g., fruit, seed, anther, flower,leaf, root); or otherwise regulatable (e.g., infection, light,temperature, chemical) promoter may be used. By inhibiting enzymes atone or more points in a metabolic pathway or genes involved inpathogenesis, the effect may be enhanced: each activity will be affectedand the effects may be magnified by targeting multiple differentcomponents. Metabolism may also be manipulated by inhibiting feedbackcontrol in the pathway or production of unwanted metabolic byproducts.

[0054] The present invention may be used to reduce crop destruction byother plant pathogens such as arachnids, insects, nematodes, protozoans,bacteria, or fungi. Some such plants and their pathogens are listed inIndex of Plant Diseases in the United States (U.S. Dept. of AgricultureHandbook No. 165, 1960); Distribution of Plant-Parasitic NematodeSpecies in North America (Society of Nematologists, 1985); and Fungi onPlants and Plant Products in the United States (AmericanPhytopathological Society, 1989). Insects with reduced ability to damagecrops or improved ability to prevent other destructive insects fromdamaging crops may be produced. Furthermore, some nematodes are vectorsof plant pathogens, and may be attacked by other beneficial nematodeswhich have no effect on plants. Inhibition of target gene activity couldbe used to delay or prevent entry into a particular developmental step(e.g., metamorphosis), if plant disease was associated with a particularstage of the pathogen's life cycle. Interactions between pathogens mayalso be modified by the invention to limit crop damage. For example, theability of beneficial nematodes to attack their harmful prey may beenhanced by inhibition of behavior-controlling nematode genes accordingto the invention.

[0055] Although pathogens cause disease, some of the microbes interactwith their plant host in a beneficial manner. For example, some bacteriaare involved in symbiotic relationships that fix nitrogen and some fungiproduce phytohormones. Such beneficial interactions may be promoted byusing the present invention to inhibit target gene activity in the plantand/or the microbe.

[0056] Another utility of the present invention could be a method ofidentifying gene function in an organism comprising the use ofdouble-stranded RNA to inhibit the activity of a target gene ofpreviously unknown function. Instead of the time consuming and laboriousisolation of mutants by traditional genetic screening, functionalgenomics would envision determining the function of uncharacterizedgenes by employing the invention to reduce the amount and/or alter thetiming of target gene activity. The invention could be used indetermining potential targets for pharmaceutics, understanding normaland pathological events associated with development, determiningsignaling pathways responsible for postnatal development/aging, and thelike. The increasing speed of acquiring nucleotide sequence informationfrom genomic and expressed gene sources, including total sequences forthe yeast, D. melanogaster, and C. elegans genomes, can be coupled withthe invention to determine gene function in an organism (e.g.,nematode). The preference of different organisms to use particularcodons, searching sequence databases for related gene products,correlating the linkage map of genetic traits with the physical map fromwhich the nucleotide sequences are derived, and artificial intelligencemethods may be used to define putative open reading frames from thenucleotide sequences acquired in such sequencing projects.

[0057] A simple assay would be to inhibit gene expression according tothe partial sequence available from an expressed sequence tag (EST).Functional alterations in growth, development, metabolism, diseaseresistance, or other biological processes would be indicative of thenormal role of the EST's gene product.

[0058] The ease with which RNA can be introduced into an intactcell/organism containing the target gene allows the present invention tobe used in high throughput screening (HTS). For example, duplex RNA canbe produced by an amplification reaction using primers flanking theinserts of any gene library derived from the target cell/organism.Inserts may be derived from genomic DNA or mRNA (e.g., cDNA and cRNA).Individual clones from the library can be replicated and then isolatedin separate reactions, but preferably the library is maintained inindividual reaction vessels (e.g., a 96-well microtiter plate) tominimize the number of steps required to practice the invention and toallow automation of the process. Solutions containing duplex RNAs thatare capable of inhibiting the different expressed genes can be placedinto individual wells positioned on a microtiter plate as an orderedarray, and intact cells/organisms in each well can be-assayed for anychanges or modifications in behavior or development due to inhibition oftarget gene activity. The amplified RNA can be fed directly to, injectedinto, the cell/organism containing the target gene. Alternatively, theduplex RNA can be produced by in vivo or in vitro transcription from anexpression construct used to produce the library. The construct can bereplicated as individual clones of the library and transcribed toproduce the RNA; each clone can then be fed to, or injected into, thecell/organism containing the target gene. The function of the targetgene can be assayed from the effects it has on the cell/organism whengene activity is inhibited. This screening could be amenable to smallsubjects that can be processed in large number, for example:arabidopsis, bacteria, drosophila, fungi, nematodes, viruses, zebrafish,and tissue culture cells derived from mammals.

[0059] A nematode or other organism that produces a colorimetric,fluorogenic, or luminescent signal in response to a regulated promoter(e.g., transfected with a reporter gene construct) can be assayed in anHTS format to identify DNA-binding proteins that regulate the promoter.In the assay's simplest form, inhibition of a negative regulator resultsin an increase of the signal and inhibition of a positive regulatorresults in a decrease of the signal.

[0060] If a characteristic of an organism is determined to begenetically linked to a polymorphism through RFLP or QTL analysis, thepresent invention can be used to gain insight regarding whether thatgenetic polymorphism might be directly responsible for thecharacteristic. For example, a fragment defining the geneticpolymorphism or sequences in the vicinity of such a genetic polymorphismcan be amplified to produce an RNA, the duplex RNA can be introduced tothe organism, and whether an alteration in the characteristic iscorrelated with inhibition can be determined. Of course, there may betrivial explanations for negative results with this type of assay, forexample: inhibition of the target gene causes lethality, inhibition ofthe target gene may not result in any observable alteration, thefragment contains nucleotide sequences that are not capable ofinhibiting the target gene, or the target gene's activity is redundant.

[0061] The present invention may be useful in allowing the inhibition ofessential genes. Such genes may be required for cell or organismviability at only particular stages of development or cellularcompartments. The functional equivalent of conditional mutations may beproduced by inhibiting activity of the target gene when or where it isnot required for viability. The invention allows addition of RNA atspecific times of development and locations in the organism withoutintroducing permanent mutations into the target genome.

[0062] If alternative splicing produced a family of transcripts thatwere distinguished by usage of characteristic exons, the presentinvention can target inhibition through the appropriate exons tospecifically inhibit or to distinguish among the functions of familymembers. For example, a hormone that contained an alternatively splicedtransmembrane domain may be expressed in both membrane bound andsecreted forms. Instead of isolating a nonsense mutation that terminatestranslation before the transmembrane domain, the functional consequencesof having only secreted hormone can be determined according to theinvention by targeting the exon containing the transmembrane domain andthereby inhibiting expression of membrane-bound hormone.

[0063] The present invention may be used alone or as a component of akit having at least one of the reagents necessary to carry out the invitro or in vivo introduction of RNA to test samples or subjects.Preferred components are the dsRNA and a vehicle that promotesintroduction of the dsRNA. Such a kit may also include instructions toallow a user of the kit to practice the invention.

[0064] Pesticides may include the RNA molecule itself, an expressionconstruct capable of expressing the RNA, or organisms transfected withthe expression construct. The pesticide of the present invention mayserve as an arachnicide, insecticide, nematicide, viricide, bactericide,and/or fungicide. For example, plant parts that are accessible aboveground (e.g., flowers, fruits, buds, leaves, seeds, shoots, bark, stems)may be sprayed with pesticide, the soil may be soaked with pesticide toaccess plant parts growing beneath ground level, or the pest may becontacted with pesticide directly. If pests interact with each other,the RNA may be transmitted between them. Alternatively, if inhibition ofthe target gene results in a beneficial effect on plant growth ordevelopment, the aforementioned RNA, expression construct, ortransfected organism may be considered a nutritional agent. In eithercase, genetic engineering of the plant is not required to achieve theobjectives of the invention.

[0065] Alternatively, an organism may be engineered to produce dsRNAwhich produces commercially or medically beneficial results, forexample, resistance to a pathogen or its pathogenic effects, improvedgrowth, or novel developmental patterns.

[0066] Used as either an pesticide or nutrient, a formulation of thepresent invention may be delivered to the end user in dry or liquidform: for example, as a dust, granulate, emulsion, paste, solution,concentrate, suspension, or encapsulation. Instructions for safe andeffective use may also be provided with the formulation. The formulationmight be used directly, but concentrates would require dilution bymixing with an extender provided by the formulator or the end user.Similarly, an emulsion, paste, or suspension may require the end user toperform certain preparation steps before application. The formulationmay include a combination of chemical additives known in the art such assolid carriers, minerals, solvents, dispersants, surfactants,emulsifiers, tackifiers, binders, and other adjuvants. Preservatives andstabilizers may also be added to the formulation to facilitate storage.The crop area or plant may also be treated simultaneously or separatelywith other pesticides or fertilizers. Methods of application includedusting, scattering or pouring, soaking, spraying, atomizing, andcoating. The precise physical form and chemical composition of theformulation, and its method of application, would be chosen to promotethe objectives of the invention and in accordance with prevailingcircumstances. Expression constructs and transfected hosts capable ofreplication may also promote the persistence and/or spread of theformulation.

[0067] Description of the dsRNA Inhibition Phenomenon in C. elegans

[0068] The operation of the present invention was shown in the modelgenetic organism Caenorhabditis elegans.

[0069] Introduction of RNA into cells had been seen in certainbiological systems to interfere with function of an endogenousgene^(1,2). Many such effects were believed to result from a simpleantisense mechanism dependent on hybridization between injectedsingle-stranded RNA and endogenous transcripts. In other cases, a morecomplex mechanism had been suggested. One instance of an RNA-mediatedmechanism was RNA interference (RNAi) phenomenon in the nematode C.elegans. RNAi had been used in a variety of studies to manipulate geneexpression^(3,4).

[0070] Despite the usefulness of RNAi in C. elegans, many features hadbeen difficult to explain. Also, the lack of a clear understanding ofthe critical requirements for interfering RNA led to a sporadic recordof failure and partial success in attempts to extend RNAi beyond theearliest stages following injection. A statement frequently made in theliterature was that sense and antisense RNA preparations are eachsufficient to cause interference^(3,4). The only precedent for such asituation was in plants where the process of co-suppression had asimilar history of usefulness in certain cases, failure in others, andno ability to design interference protocols with a high chance ofsuccess. Working with C. elegans, we discovered an RNA structure thatwould give effective and uniform genetic inhibition. The prior art didnot teach or suggest that RNA structure was a critical feature forinhibition of gene expression. Indeed the ability of crude sense andantisense preparations to produce interference^(3,4) had been taken asan indication that RNA structure was not a critical factor. Instead, theextensive plant literature and much of the ongoing research in C.elegans was focused on the possibility that detailed features of thetarget gene sequence or its chromosomal locale was the critical featurefor interfering with gene expression.

[0071] The inventors carefully purified sense or antisense RNA forunc-22 and tested each for gene-specific inhibition. While the crudesense and antisense preparations had strong interfering activity, it wasfound that the purified sense and antisense RNAs had only marginalinhibitory activity. This was unexpected because many techniques inmolecular biology are based on the assumption that RNA produced withspecific in vitro promoters (e.g., T3 or T7 RNA polymerase), or withcharacterized promoters in vivo, is produced predominantly from a singlestrand. The inventors had carried out purification of these crudepreparations to investigate whether a small fraction of the RNA had anunusual structure which might be responsible for the observed geneticinhibition. To rigorously test whether double-stranded character mightcontribute to genetic inhibition, the inventors carried out additionalpurification of single-stranded RNAs and compared inhibitory activitiesof individual strands with that of the double-stranded hybrid.

[0072] The following examples are meant to be illustrative of thepresent invention; however, the practice of the invention is not limitedor restricted in any way by them.

[0073] Analysis of RNA-Mediated Inhibition of C. elegans Genes

[0074] The unc-22 gene was chosen for initial comparisons of activity asa result of previous genetic analysis that yields a semi-quantitativecomparison between unc-22 gene activity and the movement phenotypes ofanimals^(3,8) : decreases in activity produce an increasingly severetwitching phenotype, while complete loss of function results in theadditional appearance of muscle structural defects and impairedmotility. unc-22 encodes an abundant but non-essential myofilamentprotein⁷⁻⁹. unc-22 mRNA is present at several thousand copies perstriated muscle cell³.

[0075] Purified antisense and sense RNAs covering a 742 nt segment ofunc-22 had only marginal inhibitory activity, requiring a very high doseof injected RNA for any observable effect (FIG. 4). By contrast, asense+antisense mixture produced a highly effective inhibition ofendogenous gene activity (FIG. 4). The mixture was at least two ordersof magnitude more effective than either single strand in inhibiting geneexpression. The lowest dose of the sense+antisense mixture tested,approximately 60,000 molecules of each strand per adult, led totwitching phenotypes in an average of 100 progeny. unc-22 expressionbegins in embryos with approximately 500 cells. At this point, theoriginal injected material would be diluted to at most a few moleculesper cell.

[0076] The potent inhibitory activity of the sense+antisense mixturecould reflect formation of double-stranded RNA (dsRNA), or conceivablysome alternate synergy between the strands. Electrophoretic analysisindicated that the injected material was predominantly double stranded.The dsRNA was gel purified from the annealed mixture and found to retainpotent inhibitory activity. Although annealing prior to injection wascompatible with inhibition, it was not necessary. Mixing of sense andantisense RNAs in low salt (under conditions of minimal dsRNAformation), or rapid sequential injection of sense and antisensestrands, were sufficient to allow complete inhibition. A long interval(>1 hour) between sequential injections of sense and antisense RNAresulted in a dramatic decrease in inhibitory activity. This suggeststhat injected single strands may be degraded or otherwise renderedinaccessible in the absence of the complementary strand.

[0077] An issue of specificity arises when considering known cellularresponses to dsRNA. Some organisms have a dsRNA-dependent protein kinasethat activates a panic response mechanism¹⁰. Conceivably, the inventivesense+antisense synergy could reflect a non-specific potentiation ofantisense effects by such a panic mechanism. This was not found to bethe case: co-injection of dsRNA segments unrelated to unc-22 did notpotentiate the ability of unc-22 single strands to mediate inhibition.Also investigated was whether double-stranded structure could potentiateinhibitory activity when placed in cis to a single-stranded segment. Nosuch potentiation was seen; unrelated double-stranded sequences located5′ or 3′ of a single-stranded unc-22 segment did not stimulateinhibition. Thus potentiation of gene-specific inhibition was observedonly when dsRNA sequences exist within the region of homology with thetarget gene.

[0078] The phenotype produced by unc-22 dsRNA was specific. Progeny ofinjected animals exhibited behavior indistinguishable fromcharacteristic unc-22 loss of function mutants. Target-specificity ofdsRNA effects using three additional genes with well characterizedphenotypes (FIG. 1 and Table 1). unc-54 encodes a body wall musclemyosin heavy chain isoform required for full musclecontraction^(7,11,12), fem-1 encodes an ankyrin-repeat containingprotein required in hermaphrodites for sperm production^(13,14), andhlh-1 encodes a C. elegans homolog of the myoD family required forproper body shape and motility^(15,16). For each of these genes,injection of dsRNA produced progeny broods exhibiting the known nullmutant phenotype, while the purified single strands produced nosignificant reduction in gene expression. With one exception, all of thephenotypic consequences of dsRNA injection were those expected frominhibition of the corresponding gene. The exception (segment unc54C,which led to an embryonic and larval arrest phenotype not seen withunc-54 null mutants) was illustrative. This segment covers the highlyconserved myosin motor domain, and might have been expected to inhibitthe activity of other highly related myosin heavy chain genes¹⁷. Thisinterpretation would support uses of the present invention in whichnucleotide sequence comparison of dsRNA and target gene show less than100% identity. The unc54C segment has been unique in our overallexperience to date: effects of 18 other dsRNA segments have all beenlimited to those expected from characterized null mutants.

[0079] The strong phenotypes seen following dsRNA injection areindicative of inhibitory effects occurring in a high fraction of cells.The unc-54 and hlh-1 muscle phenotypes, in particular, are known toresult from a large number of defective muscle cells^(11,16). To examineinhibitory effects of dsRNA on a cellular level, a transgenic lineexpressing two different GFP-derived fluorescent reporter proteins inbody muscle was used. Injection of dsRNA directed to gfp produceddramatic decreases in the fraction of fluorescent cells (FIG. 2). Bothreporter proteins were absent from the negative cells, while the fewpositive cells generally expressed both GFP forms.

[0080] The pattern of mosaicism observed with gfp inhibition was notrandom. At low doses of dsRNA, the inventors saw frequent inhibition inthe embryonically-derived muscle cells present when the animal hatched.The inhibitory effect in these differentiated cells persisted throughlarval growth: these cells produced little or no additional GFP as theaffected animals grew. The 14 postembryonically-derived striated musclesare born during early larval stages and were more resistant toinhibition. These cells have come through additional divisions (13-14versus 8-9 for embryonic muscles^(18,19)). At high concentrations of gfpdsRNA, inhibition was noted in virtually all striated bodywall muscles,with occasional single escaping cells including cells born in embryonicor post-embryonic stages. The nonstriated vulval muscles, born duringlate larval development, appeared resistant to genetic inhibition at alltested concentrations of injected RNA. The latter result is importantfor evaluating the use of the present invention in other systems. First,it indicates that failure in one set of cells from an organism does notnecessarily indicate complete non-applicability of the invention to thatorganism. Second, it is important to realize that not all tissues in theorganism need to be affected for the invention to be used in anorganism. This may serve as an advantage in some situations.

[0081] A few observations serve to clarify the nature of possibletargets and mechanisms for RNA-mediated genetic inhibition in C.elegans:

[0082] First, dsRNA segments corresponding to a variety of intron andpromoter sequences did not produce detectable inhibition (Table 1).Although consistent with possible inhibition at a post-transcriptionallevel, these experiments do not rule out inhibition at the level of thegene.

[0083] Second, dsRNA injection produced a dramatic decrease in the levelof the endogenous mRNA transcript (FIG. 3). Here, a mex-3 transcriptthat is abundant in the gonad and early embryos²⁰ was targeted, wherestraightforward in situ hybridization can be performed⁵. No endogenousmex-3 mRNA was observed in animals injected with a dsRNA segment derivedfrom mex-3 (FIG. 3D), but injection of purified mex-3 antisense RNAresulted in animals that retained substantial endogenous mRNA levels(FIG. 3C).

[0084] Third, dsRNA-mediated inhibition showed a surprising ability tocross cellular boundaries. Injection of dsRNA for unc-22, gfp, or lacZinto the body cavity of the head or tail produced a specific and robustinhibition of gene expression in the progeny brood (Table 2). Inhibitionwas seen in the progeny of both gonad arms, ruling out a transient“nicking” of the gonad in these injections. dsRNA injected into bodycavity or gonad of young adults also produced gene-specific inhibitionin somatic tissues of the injected animal (Table 2).

[0085] Table 3 shows that C. elegans can respond in a gene-specificmanner to dsRNA encountered in the environment. Bacteria are a naturalfood source for C. elegans. The bacteria are ingested, ground in theanimal's pharynx, and the bacterial contents taken up in the gut. Theresults show that E. coli bacteria expressing dsRNAs can confer specificinhibitory effects on C. elegans nematode larvae that feed on them.

[0086] Three C. elegans genes were analyzed. For each gene,corresponding dsRNA was expressed in E. coli by inserting a segment ofthe coding region into a plasmid construct designed for bidirectionaltranscription by bacteriophage T7 RNA polymerase. The dsRNA segmentsused for these experiments were the same as those used in previousmicroinjection experiments (see FIG. 1). The effects resulting fromfeeding these bacteria to C. elegans were compared to the effectsachieved by microinjecting animals with dsRNA.

[0087] The C. elegans gene unc-22 encodes an abundant muscle filamentprotein. unc-22 null mutations produce a characteristic and uniformtwitching phenotype in which the animals can sustain only transientmuscle contraction. When wild-type animals were fed bacteria expressinga dsRNA segment from unc-22, a high fraction (85%) exhibited a weak butstill distinct twitching phenotype characteristic of partial loss offunction for the unc-22 gene. The C. elegans fem-I gene encodes a latecomponent of the sex determination pathway. Null mutations prevent theproduction of sperm and lead euploid (XX) animals to develop as females,while wild type XX animals develop as hermaphrodites. When wild-typeanimals were fed bacteria expressing dsRNA corresponding to fem-1, afraction (43%) exhibit a sperm-less (female) phenotype and were sterile.Finally, the ability to inhibit gene expression of a transgene targetwas assessed. When animals carrying a gfp transgene were fed bacteriaexpressing dsRNA corresponding to the gfp reporter, an obvious decreasein the overall level of GFP fluorescence was observed, again inapproximately 12% of the population (see FIG. 5, panels B and C).

[0088] The effects of these ingested RNAs were specific. Bacteriacarrying different dsRNAs from fem-1 and gfp produced no twitching,dsRNAs from unc-22 and fem-1 did not reduce gfp expression, and dsRNAsfrom gfp and unc-22 did not produce females. These inhibitory effectswere apparently mediated by dsRNA: bacteria expressing only the sense orantisense strand for either gfp or unc-22 caused no evident phenotypiceffects on their C. elegans predators.

[0089] Table 4 shows the effects of bathing C. elegans in a solutioncontaining dsRNA. Larvae were bathed for 24 hours in solutions of theindicated dsRNAs (1 mg/ml), then allowed to recover in normal media andallowed to grow under standard conditions for two days. The unc-22 dsRNAwas segment ds-unc22A from FIG. 1. pos-1 and sqt-3 dsRNAs were from thefull length cDNA clones. pos-1 encodes an essential maternally providedcomponent required early in embyogenesis. Mutations removing pos-1activity have an early embryonic arrest characteristic of skn-likemutations^(29,30). Cloning and activity patterns for sqt-³ have beendescribed³¹ . C. elegans sqt-3 mutants have mutations in the col-1collagen gene³¹. Phenotypes of affected animals are noted. Incidences ofclear phenotypic effects in these experiments were 5-10% for unc-22, 50%for pos-1, and 5% for sqt-3. These are frequencies of unambiguousphenocopies; other treated animals may have had marginal defectscorresponding to the target gene that were not observable. Eachtreatment was fully gene-specific in that unc-22 dsRNA produced onlyUnc-22 phenotypes, pos-1 dsRNA produced only Pos-1 phenotypes, and sqt-3dsRNA produced only Sqt-3 phenotypes.

[0090] Some of the results described herein were published after thefiling of our provisional application. Those publications and a reviewcan be cited as Fire, A., et al. Nature, 391, 806-811, 1998; Timmons, L.& Fire, A. Nature, 395, 854, 1998; and Montgomery, M. K. & Fire, A.Trends in Genetics, 14, 255-258, 1998.

[0091] The effects described herein significantly augment availabletools for studying gene function in C. elegans and other organisms. Inparticular, functional analysis should now be possible for a largenumber of interesting coding regions²¹ for which no specific functionhave been defined. Several of these observations show the properties ofdsRNA that may affect the design of processes for inhibition of geneexpression. For example, one case was observed in which a nucleotidesequence shared between several myosin genes may inhibit gene expressionof several members of a related gene family.

[0092] Methods of RNA Synthesis and Microinjection

[0093] RNA was synthesized from phagemid clones with T3 and T7 RNApolymerase⁶, followed by template removal with two sequential DNasetreatments. In cases where sense, antisense, and mixed RNA populationswere to be compared, RNAs were further purified by electrophoresis onlow-gelling-temperature agarose. Gel-purified products appeared to lackmany of the minor bands seen in the original “sense” and “antisense”preparations. Nonetheless, RNA species accounting for less than 10% ofpurified RNA preparations would not have been observed. Without gelpurification, the “sense” and “antisense” preparations producedsignificant inhibition. This inhibitory activity was reduced oreliminated upon gel purification. By contrast, sense+antisense mixturesof gel purified and non-gel-purified RNA preparations produced identicaleffects.

[0094] Following a short (5 minute) treatment at 68° C. to removesecondary structure, sense+antisense annealing was carried out ininjection buffer²⁷ at 37° C. for 10-30 minutes. Formation ofpredominantly double stranded material was confirmed by testingmigration on a standard (non-denaturing) agarose gel: for each RNA pair,gel mobility was shifted to that expected for double-stranded RNA of theappropriate length. Co-incubation of the two strands in a low-saltbuffer (5 mM Tris-HCl pH 7.5, 0.5 mM EDTA) was insufficient for visibleformation of double-stranded RNA in vitro. Non-annealed sense+antisenseRNAs for unc22B and gfpG were tested for inhibitory effect and found tobe much more active than the individual single strands, but 2-4 foldless active than equivalent pre-annealed preparations.

[0095] After pre-annealing of the single strands for unc22A, the singleelectrophoretic species corresponding in size to that expected for dsRNAwas purified using two rounds of gel electrophoresis. This materialretained a high degree of inhibitory activity.

[0096] Except where noted, injection mixes were constructed so animalswould receive an average of 0.5×10⁶ to 1.0×10⁶ molecules of RNA. Forcomparisons of sense, antisense, and dsRNA activities, injections werecompared with equal masses of RNA (i.e., dsRNA at half the molarconcentration of the single strands). Numbers of molecules injected peradult are given as rough approximations based on concentration of RNA inthe injected material (estimated from ethidium bromide staining) andinjection volume (estimated from visible displacement at the site ofinjection). A variability of several-fold in injection volume betweenindividual animals is possible; however, such variability would notaffect any of the conclusions drawn herein.

[0097] Methods for Analysis of Phenotypes

[0098] Inhibition of endogenous genes was generally assayed in a wildtype genetic background (N2). Features analyzed included movement,feeding, hatching, body shape, sexual identity, and fertility.Inhibition with gfp²⁷ and lacZ activity was assessed using strainPD4251. This strain is a stable transgenic strain containing anintegrated array (ccIs4251) made up of three plasmids: pSAK4 (myo-3promoter driving mitochondrially targeted GFP), pSAK2 (myo-3 promoterdriving a nuclear targeted GFP-LacZ fusion), and a dpy-20 subclone²⁶ asa selectable marker. This strain produces GFP in all body muscles, witha combination of mitochondrial and nuclear localization. The twodistinct compartments are easily distinguished in these cells, allowinga facile distinction between cells expressing both, either, or neitherof the original GFP constructs.

[0099] Gonadal injection was performed by inserting the microinjectionneedle into the gonadal syncitium of adults and expelling 20-100 pl ofsolution (see Reference 25). Body cavity injections followed a similarprocedure, with needle insertion into regions of the head and tailbeyond the positions of the two gonad arms. Injection into the cytoplasmof intestinal cells was another effective means of RNA delivery, and maybe the least disruptive to the animal. After recovery and transfer tostandard solid media, injected animals were transferred to fresh cultureplates at 16 hour intervals. This yields a series of semi-synchronouscohorts in which it was straightforward to identify phenotypicdifferences. A characteristic temporal pattern of phenotypic severity isobserved among progeny. First, there is a short “clearance” interval inwhich unaffected progeny are produced. These include impermeablefertilized eggs present at the time of injection. After the clearanceperiod, individuals are produced which show the inhibitory phenotype.After injected animals have produced eggs for several days, gonads canin some cases “revert” to produce incompletely affected orphenotypically normal progeny.

[0100] Additional Description of the Results

[0101]FIG. 1 shows genes used to study RNA-mediated genetic inhibitionin C. elegans. Intron-exon structure for genes used to test RNA-mediatedinhibition are shown (exons: filled boxes; introns: open boxes; 5′ and3′ untranslated regions: shaded; sequence references are as follows:unc-22⁹, unc-54¹², fem-1¹⁴, and hlh-1¹⁵). These genes were chosen basedon: (1) a defined molecular structure, (2) classical genetic datashowing the nature of the null phenotype. Each segment tested forinhibitory effects is designated with the name of the gene followed by asingle letter (e.g., unc22C). Segments derived from genomic DNA areshown above the gene, segments derived from cDNA are shown below thegene. The consequences of injecting double-stranded RNA segments foreach of these genes is described in Table 1. dsRNA sequences from thecoding region of each gene produced a phenotype resembling the nullphenotype for that gene.

[0102] The effects of inhibitory RNA were analyzed in individual cells(FIG. 2, panels A-H). These experiments were carried out in a reporterstrain (called PD4251) expressing two different reporter proteins:nuclear GFP-LacZ and mitochondrial GFP, both expressed in body muscle.The fluorescent nature of these reporter proteins allowed us to examineindividual cells under the fluorescence microscope to determine theextent and generality of the observed inhibition of gene. ds-unc22A RNAwas injected as a negative control. GFP expression in progeny of theseinjected animals was not affected. The GFP patterns of these progenyappeared identical to the parent strain, with prominent fluorescence innuclei (the nuclear localized GFP-LacZ) and mitochondria (themitochondrially targeted GFP): young larva (FIG. 2A), adult (FIG. 2B),and adult body wall at high magnification (FIG. 2C).

[0103] In contrast, the progeny of animals injected with ds-gfpG RNA areaffected (FIGS. 2D-F). Observable GFP fluorescence is completely absentin over 95% of the cells. Few active cells were seen in larvae (FIG. 2Dshows a larva with one active cell; uninjected controls show GFPactivity in all 81 body wall muscle cells). Inhibition was not effectivein all tissues: the entire vulval musculature expressed active GFP in anadult animal (FIG. 2E). Rare GFP positive body wall muscle cells werealso seen adult animals (two active cells are shown in FIG. 2F).Inhibition was target specific (FIGS. 2G-I). Animals were injected withds-lacZL RNA, which should affect the nuclear but not the mitochondrialreporter construct. In the animals derived from this injection,mitochondrial-targeted GFP appeared unaffected while thenuclear-targeted GFP-LacZ was absent from almost all cells (larva inFIG. 2G). A typical adult lacked nuclear GFP-LacZ in almost allbody-wall muscles but retained activity in vulval muscles (FIG. 2H).Scale bars in FIG. 2 are 20 μm.

[0104] The effects of double-stranded RNA corresponding to mex-3 onlevels of the endogenous mRNA was shown by in situ hybridization toembryos (FIG. 3, panels A-D). The 1262 nt mex-3 cDNA clone²⁰ was dividedinto two segments, mex-3A and mex-3B with a short (325 nt) overlap.Similar results were obtained in experiments with no overlap betweeninhibiting and probe segments. mex-3B antisense or dsRNA was injectedinto the gonads of adult animals, which were maintained under standardculture conditions for 24 hours before fixation and in situhybridization (see Reference 5). The mex-3B dsRNA produced 100%embryonic arrest, while >90% of embryos from the antisense injectionshatched. Antisense probes corresponding to mex-3A were used to assaydistribution of the endogenous mex-3 mRNA (dark stain). Four-cell stageembryos were assayed; similar results were observed from the 1 to 8 cellstage and in the germline of injected adults. The negative control (theabsence of hybridization probe) showed a lack of staining (FIG. 3A).Embryos from uninjected parents showed a normal pattern of endogenousmex-3 RNA (FIG. 3B). The observed pattern of mex-3 RNA was as previouslydescribed in Reference 20. Injection of purified mex-3B antisense RNAproduced at most a modest effect: the resulting embryos retained mex-3mRNA, although levels may have been somewhat less than wild type (FIG.3C). In contrast, no mex-3 RNA was detected in embryos from parentsinjected with dsRNA corresponding to mex-3B (FIG. 3D). The scale of FIG.3 is such that each embryo is approximately 50 μm in length.

[0105] Gene-specific inhibitory activity by unc-22A RNA was measured asa function of RNA structure and concentration (FIG. 4). Purifiedantisense and sense RNA from unc22A were injected individually or as anannealed mixture. “Control” was an unrelated dsRNA (gfpG). Injectedanimals were transferred to fresh culture plates 6 hours (columnslabeled 1), 15 hours (columns labeled 2), 27 hours (columns labeled 3),41 hours (columns labeled 4), and 56 hours (columns labeled 5) afterinjection. Progeny grown to adulthood were scored for movement in theirgrowth environment, then examined in 0.5 mM levamisole. The main graphindicates fractions in each behavioral class. Embryos in the uterus andalready covered with an eggshell at the time of injection were notaffected and, thus, are not included in the graph. The bottom-leftdiagram shows the genetically derived relationship between unc-22 genedosage and behavior based on analyses of unc-22 heterozygotes andpolyploids^(8,3).

[0106] FIGS. 5 A-C show a process and examples of genetic inhibitionfollowing ingestion by C. elegans of dsRNAs from expressing bacteria. Ageneral strategy for production of dsRNA is to clone segments ofinterest between flanking copies of the bacteriophage T7 promoter into abacterial plasmid construct (FIG. 5A). A bacterial strain (BL21/DE3)²⁸expressing the T7 polymerase gene from an inducible (Lac) promoter wasused as a host. A nuclease-resistant dsRNA was detected in lysates oftransfected bacteria. Comparable inhibition results were obtained withthe two bacterial expression systems. A GFP-expressing C. elegansstrain, PD4251 (see FIG. 2), was fed on a native bacterial host. Theseanimals show a uniformly high level of GFP fluorescence in body muscles(FIG. 5B). PD4251 animals were also reared on a diet of bacteriaexpressing dsRNA corresponding to the coding region for gfp. Under theconditions of this experiment, 12% of these animals showed dramaticdecreases in GFP (FIG. 5C). As an alternative strategy, single copies ofthe T7 promoter were used to drive expression of an inverted-duplicationfor a segment of the target gene, either unc-22 or gfp. This wascomparably effective.

[0107] All references (e.g., books, articles, applications, and patents)cited in this specification are indicative of the level of skill in theart and their disclosures are incorporated herein in their entirety.

[0108] 1. Izant, J. & Weintraub, H. Cell 36, 1007-1015 (1984).

[0109] 2. Nellen, W. & Lichtenstein, C. TIBS 18, 419-423 (1993).

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[0113] 6. Ausubel, F., et al. Current Protocols in Molecular Biology,John Wiley N.Y. (1990).

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[0131] 24. Latham, K. Trends in Genetics 12,134-138 (1996).

[0132] 25. Mello, C. & Fire, A. Methods in Cell Biology 48, 451-482(1995).

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[0135] 28. Studier, F., et al. Methods in Enzymology 185, 60-89 (1990).

[0136] 29. Bowerman, B., et al. Cell 68, 1061-1075 (1992).

[0137] 30. Mello, C. C., et al. Cell 70, 163-176 (1992).

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[0139] 32. Goeddel, D. V. Gene Expression Technology, Academic Press,1990.

[0140] 33. Kriegler, M. Gene Transfer and Expression, Stockton Press,1990.

[0141] 34. Murray, E. J. Gene Transfer and Expression Protocols, HumanaPress, 1991. TABLE 1 Effects of sense, antisense, and mixed RNAs onprogeny of injected animals. Injected Gene and Segment Size RNA F1Phenotype unc-22 null mutants: strong unc-22 twitchers^(7,8) unc22A^(a)exon 21-22 742 sense wild type antisense wild type sense + strongtwitchers (100%) antisense unc22B exon 27 1033 sense wild type antisensewild type sense + strong twitchers (100%) antisense unc22C exon21-22^(b) 785 sense + strong twitchers (100%) antisense fem-1 fem-1 nullmutants: female (no sperm)¹³ fem1A exon 10^(c) 531 sense hermaphrodite(98%) antisense hermaphrodite (>98%) sense + female (72%) antisensefem1B intron 8 556 sense + hermaphrodite (>98%) antisense unc-54 unc-54null mutants: paralyzed^(7,11) unc54A exon 6 576 sense wild type (100%)antisense wild type (100%) sense + paralyzed (100%)^(d) antisense unc54Bexon 6 651 sense wild type (100%) antisense wild type (100%) sense +paralyzed (100%)^(d) antisense unc54C exon 1-5 1015 sense + arrestedembryos and larvae antisense (100%) unc54D promoter 567 sense + wildtype (100%) antisense unc54E intron 1 369 sense + wild type (100%)antisense unc54F intron 3 386 sense + wild type (100%) antisense hlh-1null mutants: lumpy-dumpy hlh-1 larvae¹⁶ hlh1A exons 1-6 1033 sense wildtype (<2% lpy-dpy) antisense wild type (<2% lpy-dpy) sense + lpy-dpylarvae (>90%)^(e) antisense hlh1B exons 1-2 438 sense + lpy-dpy larvae(>80%)^(e) antisense hlh1C exons 4-6 299 sense + lpy-dpy larvae(>80%)^(e) antisense hlh1D intron 1 697 sense + wild type (<2% lpy-dpy)antisense myo-3 driven GFP transgenes^(f) myo-3::NLS::gfp::lacZ makesnuclear GFP in body muscle gfpG exons 2-5 730 sense nuclear GFP-LacZpattern of parent strain antisense nuclear GFP-LacZ pattern of parentstrain sense + nuclear GFP-LacZ absent in antisense 98% of cells lacZLexon 12-14 830 sense + nuclear GFP-LacZ absent in antisense >95% ofcells makes mitochondrial GFP in body myo-3::MtLS::gfp muscle gfpG exons2-5 730 sense mitochondrial GFP pattern of parent strain antisensemitochondrial GFP pattern of parent strain sense + mitochondrial GFPabsent antisense in 98% of cells lacZL exon 12-14 830 sense +mitochondrial GFP pattern antisense of parent strain

[0142] Legend of Table 1

[0143] Each RNA was injected into 6-10 adult hermaphrodites (0.5-1×10⁶molecules into each gonad arm). After 4-6 hours (to clear pre-fertilizedeggs from the uterus) injected animals were transferred and eggscollected for 20-22 hours. Progeny phenotypes were scored upon hatchingand subsequently at 12-24 hour intervals.

[0144] a: To obtain a semi-quantitative assessment of the relationshipbetween RNA dose and phenotypic response, we injected each unc22A RNApreparation at a series of different concentrations. At the highest dosetested (3.6×10⁶ molecules per gonad), the individual sense and antisenseunc22A preparations produced some visible twitching (1% and 11% ofprogeny respectively). Comparable doses of ds-unc22A RNA producedvisible twitching in all progeny, while a 120-fold lower dose ofds-unc22A RNA produced visible twitching in 30% of progeny.

[0145] b: unc22C also carries the intervening intron (43 nt).

[0146] c: fem1A also carries a portion (131 nt) of intron 10.

[0147] d: Animals in the first affected broods (laid at 4-24 hours afterinjection) showed movement defects indistinguishable from those of nullmutants in unc-54. A variable fraction of these animals (25-75%) failedto lay eggs (another phenotype of unc-54 null mutants), while theremainder of the paralyzed animals were egg-laying positive. This mayindicate partial inhibition of unc-54 activity in vulval muscles.Animals from later broods frequently exhibit a distinct partialloss-of-function phenotype, with contractility in a subset of body wallmuscles.

[0148] e: Phenotypes of hlh-1 inhibitory RNA include arrested embryosand partially elongated L1 larvae (the hlh-1 null phenotype) seen invirtually all progeny from injection of ds-hlh1A and about half of theaffected animals from ds-hlh1B and ds-hlh1C) and a set of less severedefects (seen with the remainder of the animals from ds-hlh1B andds-hlh1C). The less severe phenotypes are characteristic of partial lossof function for hlh-1.

[0149] f: The host for these injections, PD4251, expresses bothmitochondrial GFP and nuclear GFP-LacZ. This allows simultaneous assayfor inhibition of gfp (loss of all fluorescence) and lacZ (loss ofnuclear fluorescence). The table describes scoring of animals as L1larvae. ds-gfpG caused a loss of GFP in all but 0-3 of the 85 bodymuscles in these larvae. As these animals mature to adults, GFP activitywas seen in 0-5 additional bodywall muscles and in the eight vulvalmuscles. TABLE 2 Effect of injection point on genetic inhibition ininjected animals and their progeny. Site of Injected animal dsRNAinjection phenotype Progeny Phenotype None gonad or body no twitching notwitching cavity None gonad or body strong nuclear & strong nuclear &cavity mitochondrial mitochondrial GFP GFP unc22B Gonad weak twitchersstrong twitchers unc22B Body Cavity weak twitchers strong twitchers Headunc22B Body Cavity weak twitchers strong twitchers Tail gfpG Gonad lowernuclear & rare or absent nuclear & mitochondrial mitochondrial GFP GFPgfpG Body Cavity lower nuclear & rare or absent nuclear & Tailmitochondrial mitochondrial GFP GFP lacZL Gonad lower nuclear GFP rareor absent nuclear GFP lacZL Body Cavity lower nuclear GFP rare or absentnuclear Tail GFP

[0150] TABLE 3 C. elegans can respond in a gene-specific manner toenvironmental dsRNA. Germline Bacterial Food Movement PhenotypeGFP-Transgene Expression BL21(DE3)  0% twitch <1% female <1% faint GFPBL21(DE3)  0% twitch 43% female <1% faint GFP [fem-1 dsRNA] BL21(DE3)85% twitch <1% female <1% faint GFP [unc22 dsRNA] BL21(DE3)  0% twitch<1% female 12% faint GFP [gfp dsRNA]

[0151] TABLE 4 Effects of bathing C. elegans in a solution containingdsRNA. dsRNA Biological Effect unc-22 Twitching (similar to partial lossof unc-22 function) pos-1 Embryonic arrest (similar to loss of pos-1function) sqt-3 Shortened body (Dpy) (similar to partial loss of sqt-3function)

[0152] In Table 2, gonad injections were carried out into the GFPreporter strain PD4251, which expresses both mitochondrial GFP andnuclear GFP-LacZ. This allowed simultaneous assay of inhibition with gfp(fainter overall fluorescence), lacZ (loss of nuclear fluorescence), andunc-22 (twitching). Body cavity injections were carried out into thetail region, to minimize accidental injection of the gonad; equivalentresults have been observed with injections into the anterior region ofthe body cavity. An equivalent set of injections was also performed intoa single gonad arm. For all sites of injection, the entire progeny broodshowed phenotypes identical to those described in Table 1. This includedprogeny produced from both injected and uninjected gonad arms. Injectedanimals were scored three days after recovery and showed somewhat lessdramatic phenotypes than their progeny. This could in part be due to thepersistence of products already present in the injected adult. Afterds-unc22B injection, a fraction of the injected animals twitch weaklyunder standard growth conditions (10 out of 21 animals). Levamisoletreatment led to twitching of 100% (21/21) of these animals. Similareffects were seen with ds-unc22A. Injections of ds-gfpG or ds-lacZLproduced a dramatic decrease (but not elimination) of the correspondingGFP reporters. In some cases, isolated cells or parts of animalsretained strong GFP activity. These were most frequently seen in theanterior region and around the vulva. Injections of ds-gfpG and ds-lacZLproduced no twitching, while injections of ds-unc22A produced no changein GFP fluorescence pattern.

[0153] While the present invention has been described in connection withwhat is presently considered to be practical and preferred embodiments,it is understood that the invention is not to be limited or restrictedto the disclosed embodiments but, on the contrary, is intended to covervarious modifications and equivalent arrangements included within thespirit and scope of the appended claims.

[0154] Thus it is to be understood that variations in the describedinvention will be obvious to those skilled in the art without departingfrom the novel aspects of the present invention and such variations areintended to come within the scope of the present invention.

We claim:
 1. A method to inhibit expression of a target gene in a cellcomprising introduction of a ribonucleic acid (RNA) into the cell in anamount sufficient to inhibit expression of the target gene, wherein theRNA comprises a double-stranded structure with an identical nucleotidesequence as compared to a portion of the target gene.
 2. The method ofclaim 1 in which the target gene is a cellular gene.
 3. The method ofclaim 1 in which the target gene is an endogenous gene.
 4. The method ofclaim 1 in which the target gene is a transgene.
 5. The method of claim1 in which the target gene is a viral gene.
 6. The method of claim 1 inwhich the cell is from an animal.
 7. The method of claim 1 in which thecell is from a plant.
 8. The method of claim 6 in which the cell is froman invertebrate animal.
 9. The method of claim 8 in which the cell isfrom a nematode.
 10. The method of claim 1 in which the identicalnucleotide sequence is at least 50 bases in length.
 11. The method ofclaim 1 in which the target gene expression is inhibited by at least10%.
 12. The method of claim 1 in which the cell is present in anorganism and inhibition of target gene expression demonstrates a loss-offunction phenotype.
 13. The method of claim 1 in which the RNA comprisesone strand which is self-complementary.
 14. The method of claim 1 inwhich the RNA comprises two separate complementary strands.
 15. Themethod of claim 14 further comprising synthesis of the two complementarystrands and initiation of RNA duplex formation outside the cell.
 16. Themethod of claim 14 further comprising synthesis of the two complementarystrands and initiation of RNA duplex formation inside the cell.
 17. Themethod of claim 1 in which the cell is present in an organism, and theRNA is introduced within a body cavity of the organism and outside thecell.
 18. The method of claim 1 in which the cell is present in anorganism and the RNA is introduced by extracellular injection into theorganism.
 19. The method of claim 1 in which the cell is present in afirst organism, and the RNA is introduced to the first organism byfeeding a second, RNA-containing organism to the first organism.
 20. Themethod of claim 19 in which the second organism is engineered to producean RNA duplex.
 21. The method of claim 1 in which an expressionconstruct in the cell produces the RNA.
 22. A method to inhibitexpression of a target gene comprising: (a) providing an organismcontaining a target cell, wherein the target cell contains the targetgene and the target gene is expressed in the target cell; (b) contactinga ribonucleic acid (RNA) with the organism, wherein the RNA is comprisedof a double-stranded structure with duplexed ribonucleic acid strandsand one of the strands is able to duplex with a portion of the targetgene; and (c) introducing the RNA into the target cell, therebyinhibiting expression of the target gene.
 23. The method of claim 22 inwhich the organism is an animal.
 24. The method of claim 22 in which theorganism is a plant.
 25. The method of claim 22 in which the organism isan invertebrate animal.
 26. The method of claim 22 in which the organismis a nematode.
 27. The method of claim 26 in which a formulationcomprised of the RNA is applied on or adjacent to a plant, and diseaseassociated with nematode infection of the plant is thereby reduced. 28.The method of claim 22 in which the identical nucleotide sequence is atleast 50 nucleotides in length.
 29. The method of claim 22 in which theexpression of the target gene is inhibited by at least 10%.
 30. Themethod of claim 22 in which the RNA is introduced within a body cavityof the organism and outside the target cell.
 31. The method of claim 22in which the RNA is introduced by extracellular injection into theorganism.
 32. The method of claim 22 in which the organism is contactedwith the RNA by feeding the organism food containing the RNA.
 33. Themethod of claim 32 in which a genetically-engineered host transcribingthe RNA comprises the food.
 34. The method of claim 22 in which at leastone strand of the RNA is produced by transcription of an expressionconstruct.
 35. The method of claim 35 in which the organism is anematode and the expression construct is contained in a plant, anddisease associated with nematode infection of the plant is therebyreduced.
 36. A cell containing an expression construct, wherein theexpression construct transcribes at least one ribonucleic acid (RNA) andthe RNA forms a double-stranded structure with duplexed strands ofribonucleic acid, whereby said cell contains the double-stranded RNAstructure and is able to inhibit expression of a target gene when theRNA is contacted with an organism containing the target gene.
 37. Atransgenic animal containing said cell of claim
 36. 38. A transgenicplant containing said cell of claim
 36. 39. A kit comprising reagentsfor inhibiting expression of a target gene in a cell, wherein said kitcomprises a means for introduction of a ribonucleic acid (RNA) into thecell in an amount sufficient to inhibit expression of the target gene,and wherein the RNA has a double-stranded structure with an identicalnucleotide sequence as compared to a portion of the target gene.